Prodrugs of CC-1065 analogs

ABSTRACT

Prodrugs of analogs of the anti-tumor antibiotic CC-1065 having a cleavable protective group such as a piperazino carbamate, a 4-piperidino-piperidino carbamate or a phosphate, in which the protecting group confers enhanced water solubility and stability upon the prodrug, and in which the prodrug also has a moiety, such as a disulfide, that can conjugate to a cell binding reagent such as an antibody. The therapeutic use of such prodrug conjugates is also described; such prodrugs of cytotoxic agents have therapeutic use because they can deliver cytotoxic prodrugs to a specific cell population for enzymatic conversion to cytoxic drugs in a targeted fashion.

This is a divisional of application Ser. No. 10/692,856 filed Oct. 27,2003 now U.S. Pat. No. 7,049,316, which is a divisional of applicationSer. No. 10/116,053, filed Apr. 5, 2002 (now U.S. Pat. No. 6,756,397which issued Jun. 29, 2004). The entire disclosures of said priorapplications are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to novel prodrugs of cytotoxic agents andtheir therapeutic uses. More specifically, the invention relates tonovel prodrugs of cytotoxic agents that are analogs of CC-1065 and whichcomprise both a moiety for chemical linkage to a cell binding agent anda protecting group that is cleaved in vivo. The prodrugs can bechemically linked to cell binding agents to provide therapeutic agentscapable of being activated and released in vivo, and delivered tospecific cell populations in a targeted manner.

BACKGROUND OF THE INVENTION

Many reports have appeared which are directed to the targeting of tumorcells with monoclonal antibody-drug conjugates {Sela et al, inImmunoconjugates, pp. 189-216 (C. Vogel, ed. 1987); Ghose et al, inTargeted Drugs, pp. 1-22 (E. Goldberg, ed. 1983); Diener et al, inAntibody Mediated Delivery Systems, pp. 1-23 (J. Rodwell, ed. 1988);Pietersz et al, in Antibody Mediated Delivery Systems, pp. 25-53 (J.Rodwell, ed. 1988); Bumol et al, in Antibody Mediated Delivery Systems,pp. 55-79 (J. Rodwell, ed. 1988); G. A. Pietersz & K. Krauer, 2 J. DrugTargeting, 183-215 (1994); R. V. J. Chari, 31 Adv. Drug Delivery Revs.,89-104 (1998); W. A. Blattler & R. V. J. Chari, in Anticancer Agents,Frontiers in Cancer Chemotherapy, 317-338, ACS Symposium Series 796; andI. Ojima et al eds, American Chemical Society 2001}. Cytotoxic drugssuch as methotrexate, daunorubicin, doxorubicin, vincristine,vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin andmaytansinoids have been conjugated to a variety of murine monoclonalantibodies. In some cases, the drug molecules were linked to theantibody molecules through an intermediary carrier molecule such asserum albumin {Garnett et al, 46 Cancer Res. 2407-2412 (1986); Ohkawa etal, 23 Cancer Immunol. Immunother. 81-86 (1986); Endo et al, 47 CancerRes. 1076-1080 (1980)}, dextran {Hurwitz et al, 2 Appl. Biochem. 25-35(1980); Manabi et al, 34 Biochem. Pharmacol. 289-291 (1985); Dillman etal, 46 Cancer Res. 4886-4891 (1986); and Shoval et al, 85 Proc. Natl.Acad. Sci. U.S.A. 8276-8280 (1988)}, or polyglutamic acid {Tsukada etal, 73 J. Natl. Canc. Inst. 721-729 (1984); Kato et al, 27 J. Med. Chem.1602-1607 (1984); Tsukada et al, 52 Br. J. Cancer 111-116 (1985)}.

A wide array of linkers is now available for the preparation of suchimmunoconjugates, including both cleavable and non-cleavable linkers. Invitro cytotoxicity tests, however, have revealed that antibody-drugconjugates rarely achieve the same cytotoxic potency as the freeunconjugated drugs. This has suggested that mechanisms by which drugmolecules are released from conjugated antibodies are very inefficient.Early work in the area of immunotoxins showed that conjugates formed viadisulfide bridges between monoclonal antibodies and catalytically activeprotein toxins were more cytotoxic than conjugates containing otherlinkers {Lambert et al, 260 J. Biol. Chem. 12035-12041 (1985); Lambertet al, in Immunotoxins 175-209 (A. Frankel, ed. 1988); Ghetie et al, 48Cancer Res. 2610-2617 (1988)}. This improved cytotoxicity was attributedto the high intracellular concentration of reduced glutathionecontributing to the efficient cleavage of the disulfide bond between theantibody molecule and the toxin. Maytansinoids and calicheamicin werethe first examples of highly cytotoxic drugs that had been linked tomonoclonal antibodies via disulfide bonds. Antibody conjugates of thesedrugs have been shown to possess high potency in vitro and exceptionalantitumor activity in human tumor xenograft models in mice {R. V. J.Chari et al., 52 Cancer Res., 127-131 (1992); C. Liu et al., 93, Proc.Natl. Acad. Sci., 8618-8623 (1996); L. M. Hinman et al., 53, CancerRes., 3536-3542 (1993); and P. R. Hamann et al, 13, BioConjugate Chem.,40-46 (2002)}.

An attractive candidate for the preparation of such cytotoxic conjugatesis CC-1065, which is a potent anti-tumor antibiotic isolated from theculture broth of Streptomyces zelensis. CC-1065 is about 1000-fold morepotent in vitro than are commonly used anti-cancer drugs, such asdoxorubicin, methotrexate and vincristine {B. K. Bhuyan et al., CancerRes., 42, 3532-3537 (1982)}.

The structure of CC-1065 (Compound 1, FIG. 1A) has been determined byx-ray crystallography {Martin, D. G. et al, 33 J. Antibiotics 902-903(1980), and Chidester, C. G., et al, 103 J. Am. Chem. Soc. 7629-7635(1981)}. The CC-1065 molecule consists of 3 substituted pyrroloindolemoieties linked by amide bonds. The “A” subunit has a cyclopropyl ringcontaining the only asymmetric carbons in the molecule. While only therelative configuration of these carbons is available from x-ray data,the absolute configuration has been inferred as 3b-R, 4a-S, by using DNAas a chiral reagent {Hurley, L. H. et al, 226 Science 843-844 (1984)}.The “B” and “C” subunits of CC-1065 are identical pyrroloindolemoieties.

The cytotoxic potency of CC-1065 has been correlated with its alkylatingactivity and its DNA-binding or DNA-intercalating activity. These twoactivities reside in separate parts of the molecule. Thus, thealkylating activity is contained in the cyclopropapyrroloindole (CPI)subunit and the DNA-binding activity resides in the two pyrroloindolesubunits (FIG. 1A).

However, although CC-1065 has certain attractive features as a cytotoxicagent, it has limitations in therapeutic use. Administration of CC-1065to mice caused a delayed hepatotoxicity leading to mortality on day 50after a single intravenous dose of 12.5 μg/kg {V. L. Reynolds et al., J.Antibiotics, XXIX, 319-334 (1986)}. This has spurred efforts to developanalogs that do not cause delayed toxicity, and the synthesis of simpleranalogs modeled on CC-1065 has been described {M. A. Warpehoski et al.,J. Med. Chem., 31, 590-603 (1988)}. In another series of analogs, theCPI moiety was replaced by a cyclopropabenzindole (CBI) moiety {D. L.Boger et al., J. Org. Chem., 55, 5823-5833, (1990), D. L. Boger et al.,BioOrg. Med. Chem. Lett., 1, 115-120 (1991)}. These compounds maintainthe high in vitro potency of the parental drug, without causing delayedtoxicity in mice. Like CC-1065, these compounds are alkylating agentsthat bind to the minor groove of DNA in a covalent manner to cause celldeath. However, clinical evaluation of the most promising analogs,Adozelesin and Carzelesin, has led to disappointing results {B. F.Foster et al., Investigational New Drugs, 13, 321-326 (1996); I. Wolffet al., Clin. Cancer Res., 2, 1717-1723 (1996)}. These drugs displaypoor therapeutic effects because of their high systemic toxicity.

The therapeutic efficacy of CC-1065 analogs can be greatly improved bychanging the in vivo distribution through targeted delivery to the tumorsite, resulting in lower toxicity to non-targeted tissues, and thus,lower systemic toxicity. In order to achieve this goal, conjugates ofanalogs and derivatives of CC-1065 with cell-binding agents thatspecifically target tumor cells have been described {U.S. Pat. Nos.;5,475,092; 5,585,499; 5,846,545}. These conjugates typically displayhigh target-specific cytotoxicity in vitro, and exceptional anti-tumoractivity in human tumor xenograft models in mice {R. V. J. Chari et al.,Cancer Res., 55, 4079-4084 (1995)}.

Cell-binding agents are typically only soluble in aqueous medium, andare usually stored in aqueous solutions. Thus, these analogs shouldpossess sufficient water solubility to allow for efficient reaction withcell-binding agents and subsequent formulation in aqueous solution. Inaddition, for cell-binding agent conjugates to have a useful shelf life,it is important that CC-1065 analogs that are linked to thesecell-binding agents are stable for an extended period of time in aqueoussolutions.

The CC-1065 analogs described thus far (see, e.g. FIGS. 1B and 1C) areonly sparingly soluble in water. Because of the sparing solubility ofCC-1065 analogs, conjugation reactions with cell-binding agentscurrently have to be performed in extremely dilute aqueous solutions.Therefore, these prodrugs should have enhanced water solubility ascompared to the parent drugs.

Also, CC-1065 analogs that have been described thus far are quiteunstable in aqueous solutions for the following reason. The seco-form ofthe drug is spontaneously converted into the cyclopropyl form, whichthen may alkylate DNA, if present. However, the competing reaction ofthe cyclopropyl form with water results in opening of the cyclopropylring to yield the hydroxy compound, which is inactive. Thus, there is aneed to protect the reactive portion of CC-1065 analogs in order toextend their useful life in aqueous solution, for example by thedevelopment of prodrugs of CC-1065 analogs.

There is therefore a need to develop prodrugs of CC-1065 analogs thatare very stable upon storage in aqueous solutions. Preferably, theseprodrugs should only be converted into active drugs in vivo. Once theprodrug is infused into a patient, it should preferably be efficientlyconverted into active drug.

Carzelesin is a prodrug where the phenolic group in adozelesin isprotected as a phenyl carbamate {L. H. Li et al., Cancer Res., 52,4904-4913 (1992) }. However, this prodrug is too labile for therapeuticuse, and also affords no increase in water solubility compared to theparental drug. In a second example, the phenolic residue of a CC-1065analog was glycosylated to produce a prodrug (U.S. Pat. No. 5,646,298).However, this prodrug is not converted into active drug in vivo, andrequires the additional administration of an enzyme from a bacterialsource to convert it to the cytotoxic form.

There are a few examples of anticancer drugs, unrelated to CC-1065, thathave been converted into water soluble prodrugs. In the anticancer drugirinotecan, the phenolic group is protected by a 4-piperidino-piperidinocarbamate. It has been reported that this protecting group confers watersolubility to the drug. In addition, the prodrug is readily converted invivo in humans to the active drug, presumably by the enzymecarboxylesterase, which naturally exists in human serum, tumor tissueand in some organs {A. Sparreboom, 4, Clin. Cancer Res., 2747-2754(1998). L. P. Rivory et al., 52, Biochem Pharmacol., 1103-1111 (1996)}.

Similarly, the anticancer drug etoposide phosphate is an example of aprodrug that has a phosphate protecting group and is rapidly convertedinto active drug in vivo, presumably through hydrolysis by endogenousalkaline phosphatase {S. Z. Fields et al., 1 Clin. Cancer Res., 105-111(1995)}.

Thus, there exists a need for analogs of CC-1065 that have increasedsolubility and stability in aqueous solution, to facilitate theirconjugation to cell binding agents in aqueous solutions, whilepreserving their biological activity. In addition, in order to reducetoxic side-effects, it would be advantageous to provide the CC-1065analog in the form of a prodrug that is converted to the cytotoxic drugpredominantly at the desired therapeutic site and preferably through theaction of endogenous agents. All these advantages and more are providedby the invention described herein, as will be apparent to one of skillin the art upon reading the following disclosure and examples.

SUMMARY OF THE INVENTION

The object of the present invention is to provide prodrugs of CC-1065analogs, which have enhanced stability and solubility in aqueous medium.This and other objects have been achieved by providing prodrugs in whichthe phenolic group of the alkylating portion of the molecule isprotected with a functionality that renders the drug stable upon storagein aqueous solution. In addition, the protecting group confers increasedwater solubility to the drug compared to an unprotected analog. Theprotecting group is readily cleaved in vivo to give the correspondingactive drug. In the prodrugs described herein, the phenolic substituentis preferentially protected as a piperazino carbamate, a4-piperidino-piperidino carbamate or as a phosphate, each of whichpossesses a charge at physiological pH, and thus have enhanced watersolubility. In order to further enhance water solubility, an optionalpolyethylene glycol spacer has been introduced into the linking groupbetween the terminal indolyl subunit C and the cleavable linkage such asa disulfide group. The introduction of this spacer does not alter thepotency of the drug.

A more specific embodiment of the invention provides a prodrug thatcomprises an analog of a seco-cyclopropabenzindole-containing cytotoxicdrug that has a protecting group, which enhances water solubility andstability and that can be cleaved in vivo. The prodrug of this specificembodiment has a first and a second subunit that are linked by an amidebond from the secondary amino group of the pyrrole moiety of the firstsubunit to the C-2 carboxyl of the second subunit. The first subunit isshown as formula (I), and is conjugated to the second subunit, which isselected from among formulae (II)-(IX):

in which R represents a linking group that provides for linkage of theprodrug to a cell binding agent, where such linkage is preferably via adisulfide bond. The linking group may comprise a polyethylene glycolspacer. R₁-R₆ are each independently hydrogen, C₁-C₃ linear alkyl,methoxy, hydroxyl, primary amino, secondary amino, tertiary amino, oramido. R₇ is the protecting group that can be cleaved in vivo andenhances water solubility of the cyclopropabenzindole-containingcytotoxic drug, and is preferably a piperazino carbamate, a4-piperidino-piperidino carbamate or a phosphate.

The prodrugs of the invention can be used in cytotoxic conjugates inwhich a cell binding agent is linked to one or more of the prodrugs ofthe present invention. Cell binding agents include antibodies andfragments thereof, interferons, lymphokines, vitamins, hormones andgrowth factors. Pharmaceutical compositions containing such conjugatesare also provided.

The cytotoxic conjugates can be used in a method for treating a subjectby administering an effective amount of the above pharmaceuticalcomposition. According to the cell-type to which the selected cellbinding agent binds, many diseases may be treated either in vivo, exvivo or in vitro. Such diseases include, for example, the treatment ofmany kinds of cancers, including lymphomas, leukemias, cancer of thelung, breast, colon, prostate, kidney, pancreas, and the like.

Thus, there are provided prodrugs of CC-1065 analogs that have improvedsolubility and stability in aqueous solution, and which retaincytotoxicity when activated to produce an alkylating drug, and which areuseful in the targeting of specific cell types by means of conjugationto a specific cell binding agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the structure of CC-1065 and its subunits A, B, and C.

FIG. 1B and FIG. 1C show the structures of two known analogs of CC-1065.

FIG. 2 shows the structures of exemplary CC-1065 analogs and prodrugs ofthe present invention.

FIG. 3 shows the structures of exemplary polyethylene glycol-containingprodrugs of the present invention.

FIGS. 4A and B are synthesis schemes for preparing(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-methylpiperazino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC2) and(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-piperidino-piperidino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC3).

FIG. 5 shows schemes for the synthesis of PEGylated versions of DC1, DC2and DC3, which are DC5, DC6 and DC7, respectively.

FIG. 6 shows two synthetic schemes for the preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-(phosphonoxy)-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC4)).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have found that the stability, water solubilityand utility of certain CC-1065 analogs are enhanced by protection of thealkylating moiety of the analog with a suitable protecting group. Theinventors have thereby provided prodrugs of CC-1065 analogs havingenhanced aqueous solubility and stability and which are further capableof linkage to cell binding agents whereby the therapeutic efficacy ofsuch prodrugs of CC-1065 analogs is improved by changing the in vivodistribution through targeted delivery of the prodrug to the tumor site,resulting in a lower toxicity to non-targeted tissues, and hence lowersystemic toxicity. Upon delivery of the prodrug, endogenous substancessubstantially convert the prodrug to its active drug form, and, inembodiments having a cleavable linker to the cell binding agent, theactive drug form of the CC-1065 analog is released, thus furtherenhancing its cytotoxic activity. Alternatively, the linker to the cellbinding agent may be first cleaved inside the target cell to release theprodrug, followed by endogenous conversion into the active drug.

In order to achieve this goal, the inventors synthesized exemplaryprodrugs (FIGS. 2-6) of CC-1065 analogs that areseco-cyclopropabenzindole (CBI)-containing cytotoxic prodrugscomprising: (a) a first subunit of formula (I) that is protected at thephenolic hydroxyl by a protecting group to enhance stability and watersolubility and which is cleaved in vivo, and (b) a second subunit havingthe structure represented by one of formulae (II)-(IX) and whichcomprises a linking group for conjugation of the prodrug to a cellbinding agent. The linking group can contain a polyethylene glycolspacer (FIG. 3). Removal of the protecting group of the prodrug producesan active form of the drug that retains the high cytotoxicity of theparent drug. The linker is used for conjugation to cell binding agents,preferably via a disulfide bond.

It has previously been shown that the linkage of highly cytotoxic drugsto antibodies using a cleavable link, such as a disulfide bond, ensuresthe release of fully active drug inside the cell, and that suchconjugates are cytotoxic in an antigen specific manner {R. V. J. Chariet al, 52 Cancer Res. 127-131 (1992); R. V. J. Chari et al., 55 CancerRes. 4079-4084 (1995); and U.S. Pat. Nos. 5,208,020 and 5,475,092}. Inthe present invention, the inventors describe the synthesis of prodrugsof CC-1065 analogs, procedures for their conjugation to monoclonalantibodies and for measurement of the in vitro cytotoxicity andspecificity of such conjugates. Thus the invention provides usefulcompounds for the preparation of therapeutic agents directed to theelimination of diseased or abnormal cells that are to be killed or lysedsuch as tumor cells, virus infected cells, microorganism infected cells,parasite infected cells, autoimmune cells (cells that produceauto-antibodies), activated cells (those involved in graft rejection orgraft vs. host disease), or any other type of diseased or abnormalcells, while exhibiting minimal side effects.

Thus, this invention teaches the synthesis of prodrug analogs andderivatives of CC-1065 that can be chemically linked to a cell bindingagent and that maintain, upon release of the protective group, the highcytotoxicity of the parent compound CC-1065. Further, upon activation,these compounds when linked to a cell binding agent are cytotoxic tocells to which the cell binding agent binds and are much less toxic tonon-target cells.

Prodrugs of the Present Invention

The prodrugs according to the present invention comprise an analog ofCC-1065 in which the phenolic group of the alkylating portion of themolecule is protected and the prodrug further comprises a linker capableof conjugating the prodrug to a cell binding agent. The prodrug maycomprise a first and a second subunit that are linked via an amide bond.

According to certain embodiments of the present invention, the prodrugof the CC-1065 analog has a first subunit that is a seco-CBI(cyclopropabenzindole unit) in its open chloromethyl form, wherein thefirst subunit has a phenolic hydroxyl that is protected by awater-soluble protecting group that can be cleaved in vivo. The secondsubunit of the prodrug of certain embodiments of the present inventioncomprises an analog of the combined B and C subunits of CC-1065 (FIG. 1)that are 2-carboxy-indole or 2-carboxy-benzofuran derivatives, or both,and are represented by formulae (II)-(IX). As may be ascertained fromthe natural CC-1065 and from the properties of the analogs that havebeen published {e.g. Warpehoski et al, 31 J. Med. Chem. 590-603 (1988),Boger et al, 66 J. Org. Chem. 6654-6661 (2001)}, the B and C subunitscan also carry different substituents at different positions on theindole or benzofuran rings, corresponding to positions R₁-R₆ of formulae(II)-(IX), and still retain potent cytotoxic activity.

In order to link the prodrug of the CC-1065 analog to a cell-bindingagent, the prodrug must first include a moiety that allows thederivatives to be linked to a cell binding agent via a cleavable linkagesuch as a disulfide bond, an acid-labile group, a photo-labile group, apeptidase-labile group, or an esterase-labile group. The prodrug analogsare prepared so that they contain a moiety necessary to link the analogto a cell binding agent via, for example, a disulfide bond, anacid-labile group, a photo-labile group, a peptidase-labile group, or anesterase-labile group. In order to further enhance solubility in aqueoussolutions, the linking group can contain a polyethylene glycol spacer(FIG. 3).

Preferably, a disulfide linkage is used because the reducing environmentof the targeted cell results in cleavage of the disulfide and release ofthe prodrug (or drug, depending on the relative sequence of cleavage ofthe prodrug from the cell binding agent and hydrolysis of the protectinggroup), with an associated increase in cytotoxicity.

More specifically, according to certain embodiments of the presentinvention, the prodrug of an analog of CC-1065 comprises first andsecond subunits that are covalently linked via an amide bond from thesecondary amino group of the pyrrole moiety of the first subunit to theC-2 carboxy group of the second subunit having the formulae (II)-(IX).

Within formulae (II)-(IX), R represents a moiety that enables linkage ofthe prodrug of a CC-1065 analog to a cell binding agent. The linkingmoiety may contain a polyethylene glycol spacer. Examples includemoieties that enable linkages via disulfide bond, an acid-labile group,a photo-labile group, a peptidase-labile group, or an esterase-labilegroup, and are well-known in the art {see, e.g., U.S. Pat. No.5,846,545, which is incorporated herein by reference}. Preferredmoieties are those that enable linkage via a disulfide bond, for examplea thiol (DC1, DC2, DC3, DC4, DC5, DC6, DC7) or a disulfide (DC1-SMe,DC2-SMe, DC3-SMe, DC4-SMe, DC5-SMe, DC6-SMe, DC7-Sme, see FIGS. 2-6).Mixed disulfides containing any terminal leaving group, such asthiomethyl (DC1-SMe, DC2-SMe, DC3-SMe, DC4-SMe, DC5-SMe, DC6-SMe,DC7-SMe), glutathione, alkyl thiol, thiopyridyl, aryl thiol, and thelike may be used provided that such disulfides are capable of undergoinga disulfide-exchange reaction for the coupling of the prodrug to a cellbinding agent. R can optionally further comprise a spacer regioninterposed between the reactive group of the linkage-enabling portionand the 2-carboxy-indole or 2-carboxy-benzofuran derivative portion.Preferred embodiments include NHCO(CH₂)_(m) SZ, NHCOC₆H₄ (CH₂)_(m)SZ, orO(CH₂)_(m) SZ, NHCO(CH₂)_(m)(OCH₂CH₂)_(n) SZ, NHCOC₆H₄(CH₂)_(m)(OCH₂CH₂)_(n)SZ, or O(CH₂)_(m)(OCH₂CH₂)_(n) SZ wherein: Zrepresents H or SR₈, wherein R₈ represents methyl, linear alkyl,branched alkyl, cyclic alkyl, simple or substituted aryl orheterocyclic, and m represents an integer of 1 to 10, n represents aninteger of 4 to 1000. Examples of linear alkyls represented by R₈include methyl, ethyl, propyl, butyl, pentyl and hexyl. Examples ofbranched alkyls represented by R₈ include isopropyl, isobutyl,sec.-butyl, tert.-butyl, isopentyl and 1-ethyl-propyl. Examples ofcyclic alkyls represented by R₈ include cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl. Examples of simple aryls represented by R₈include phenyl and naphthyl. Examples of substituted aryls representedby R₈ include aryls such as phenyl or naphthyl substituted with alkylgroups, with halogens, such as Cl, Br, F, nitro groups, amino groups,sulfonic acid groups, carboxylic acid groups, hydroxy groups and alkoxygroups. Heterocyclics represented by R₈ are compounds wherein theheteroatoms are selected from O, N, and S, and examples include furyl,pyrrollyl, pyridyl, (e.g., a 2-substituted pyrimidine group) andthiophene. Most preferred embodiments of R include NHCO(CH₂)₂SH andNHCO(CH₂)₂SSCH₃. NHCO(CH₂)₂(OCH₂CH₂)_(n)SH andNHCO(CH₂)₂(OCH₂CH₂)_(n)SSCH₃.

Within formulae (II)-(IX), R₁ to R₆, which may be the same or different,independently represent hydrogen, C₁-C₃ linear alkyl, methoxy, hydroxyl,primary amino, secondary amino, tertiary amino, or amido. Examples ofprimary amino group-containing substituents are methyl amino, ethylamino, and isopropyl amino. Examples of secondary amino group-containingsubstituents are dimethyl amino, diethyl amino, and ethyl-propyl amino.Examples of tertiary amino group-containing substituents are trimethylamino, triethyl amino, and ethyl-isopropyl-methyl amino. Examples ofamido groups include N-methyl-acetamido, N-methyl-propionamido,N-acetamido, and N-propionamido.

Within formulae (II)-(IX), R₇ is an in vivo-cleavable protecting groupthat enhances water solubility of theseco-cyclopropabenzindole-containing cytotoxic drug. Examples ofpreferred in vivo-cleavable protecting groups are piperazino carbamate,a 4-piperidino-piperidino carbamate and a phosphate, and derivativesthereof. Thus, piperazino carbamate and 4-piperidino-piperidinocarbamate protecting groups are enzyme-cleavable by enzymes such ascarboxyl esterase, which occurs in serum and plasma. Phosphateprotecting groups are cleavable by phosphatase enzymes such as alkalinephosphatase.

Disulfide-containing and mercapto-containing prodrugs of CC-1065 analogsof the present invention can be evaluated for their ability to suppressproliferation of various unwanted cell lines in vitro only after theyhave been activated. For example, phosphoryl group-containing prodrugs,such as DC4, can be activated by incubation with commercially availablealkaline phosphatases, while carbamate-containing prodrugs, such as DC3and DC4, can be activated by incubation with commercially availablecarboxyl esterases. Cell lines such as, for example, the humanepidermoid carcinoma line KB, the human breast tumor line SK-BR-3, andthe Burkitt's lymphoma line Namalwa can easily be used for theassessment of the cytotoxicity of these compounds. Cells to be evaluatedcan be exposed to the compounds for 24 hours and the surviving fractionsof cells measured in direct assays by known methods. IC₅₀ values canthen be calculated from the results of the assays.

Preparation of Cell Binding Agents

The effectiveness of the prodrug compounds of the invention astherapeutic agents depends upon the careful selection of an appropriatecell binding agent. Cell binding agents may be of any kind presentlyknown, or that become known, and include peptides and non-peptides.Generally, these can be antibodies (especially monoclonal antibodies) ora fragment of an antibody that contains at least one binding site,lymphokines, hormones, growth factors, nutrient-transport molecules(such as transferrin), or any other cell binding molecule or substance.More specific examples of cell binding agents that can be used include:

-   monoclonal antibodies;-   single chain antibodies;-   fragments of antibodies such as Fab, Fab′, F(ab′)₂ and F_(v)    {Parham, 131 J. Immunol. 2895-2902 (1983); Spring et al, 113 J.    Immunol. 470-478 (1974); Nisonoff et al, 89 Arch. Biochem. Biophys.    230-244 (1960)};-   interferons;-   peptides;-   lymphokines such as IL-2, IL-3, IL-4, IL-6;-   hormones such as insulin, TRH (thyrotropin releasing hormones), MSH    (melanocyte-stimulating hormone), steroid hormones, such as    androgens and estrogens;-   growth factors and colony-stimulating factors such as EGF, TGFα,    insulin like growth factor (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF    {Burgess, 5 Immunology Today 155-158 (1984)};-   vitamins, such as folate and-   transferrin {O'Keefe et al, 260 J. Biol. Chem. 932-937 (1985)}.

Monoclonal antibody technology permits the production of extremelyselective cell binding agents in the form of specific monoclonalantibodies. Particularly well known in the art are techniques forcreating monoclonal antibodies produced by immunizing mice, rats,hamsters or any other mammal with the antigen of interest such as theintact target cell, antigens isolated from the target cell, whole virus,attenuated whole virus, and viral proteins such as viral coat proteins.

Selection of the appropriate cell binding agent is a matter of choicethat depends upon the particular cell population that is to be targeted,but in general monoclonal antibodies are preferred if an appropriate oneis available.

For example, the monoclonal antibody MY9 is a murine IgG₁ antibody thatbinds specifically to the CD33 Antigen {J. D. Griffin et al 8 LeukemiaRes., 521 (1984)} and can be used if the target cells express CD33 as inthe disease of acute myelogenous leukemia (AML). Similarly, themonoclonal antibody anti-B4 is a murine IgG₁, that binds to the CD19antigen on B cells {Nadler et al, 131 J. Immunol. 244-250 (1983)} andcan be used if the target cells are B cells or diseased cells thatexpress this antigen such as in non-Hodgkin's lymphoma or chroniclymphoblastic leukemia.

Additionally, GM-CSF which binds to myeloid cells can be used as a cellbinding agent to diseased cells from acute myelogenous leukemia. IL-2,which binds to activated T-cells, can be used for prevention oftransplant graft rejection, for therapy and prevention ofgraft-versus-host disease, and for the treatment of acute T-cellleukemia. MSH, which binds to melanocytes, can be used for the treatmentof melanoma.

Preparation of Prodrug Conjugates

Conjugates of the prodrugs and a cell binding agent can be formed usingany techniques presently known or later developed. An indolyl,benzofuranyl, bis-indolyl, bis-benzofuranyl, indolyl-benzofuranyl, orbenzofuranyl-indolyl derivative coupled to the seco-CBI analog can beprepared to contain a free amino group and then linked to an antibody orother cell binding agent via an acid labile linker, or by a photolabilelinker. The prodrug compounds can be condensed with a peptide having asuitable sequence and subsequently linked to a cell binding agent toproduce a peptidase labile linker. Cytotoxic compounds can be preparedto contain a primary hydroxyl group, which can be succinylated andlinked to a cell binding agent to produce a conjugate that can becleaved by intracellular esterases to liberate free prodrug. Preferably,the prodrug compounds are synthesized to contain a free or protectedthiol group, with or without a PEG-containing spacer, and then one ormore disulfide or thiol-containing prodrugs are each covalently linkedto the cell binding agent via a disulfide bond.

Representative conjugates of the invention are conjugates of prodrugs ofCC-1065 analogs with antibodies, antibody fragments, epidermal growthfactor (EGF), melanocyte stimulating hormone (MSH), thyroid stimulatinghormone (TSH), estrogen, estrogen analogs, androgen, and androgenanalogs.

Representative examples of the preparation of various conjugates ofprodrugs of CC-1065 analogs and cell binding agents are described below.

Disulfide linkers: Antibody N901 which binds to the CD-56 antigen thatis expressed on the surface of small cell lung cancer cells {J. D.Griffin, T. Hercend, R. Beveridge & S. F. Schlossman, J. Immunol,130:2947 (1983)) can be used for the preparation of conjugates. Theantibody is modified with N-succinimidyl-3-pyridyldithio propionate aspreviously described {J. Carlsson, H. Drevin & R. Axen, Biochem. J.,173:723 (1978)} to introduce, on the average, 4 pyridyldithio groups perantibody molecule. The modified antibody is reacted with thethiol-containing prodrug to produce a disulfide-linked conjugate.

Acid-Labile Linkers: Amino group-containing prodrugs of the presentinvention can be linked to antibodies and other cell binding agents viaan acid labile linker as previously described. {W. A. Blattler et al,Biochemistry 24, 1517-1524 (1985); U.S. Pat. Nos. 4,542,225, 4,569,789,4,618,492, 4,764,368}.

Similarly, an hydrazido group-containing prodrug of the presentinvention can be linked to the carbohydrate portion of antibodies andother cell binding agents via an acid labile hydrazone linker {forexamples of hydrazone linkers see B. C. Laguzza et al, J. Med. Chem.,32, 548-555 (1989); R. S. Greenfield et al, Cancer Res., 50, 6600-6607(1990)}.

Photo-Labile Linkers: Amine group containing prodrugs of the presentinvention may be linked to antibodies and other cell binding agents viaa photolabile linker as previously described {P. Senter et al,Photochemistry and Photobiology, 42, 231-237 (1985); U.S. Pat. No.4,625,014}.

Peptidase-Labile Linkers: Amine group containing prodrugs of the presentinvention may also be linked to cell binding agents via peptide spacers.It has been previously shown that short peptide spacers between drugsand macromolecular protein carriers are stable in serum but are readilyhydrolyzed by intracellular peptidases {A. Trouet et al, Proc. Natl.Acad. Sci., 79, 626-629 (1982)}. The amino group containing containingprodrugs may be condensed with peptides using condensing agents such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (EDC-HCl) to give apeptide derivative that can be linked to cell binding agents.

Esterase-Labile Linkers: Prodrugs of the present invention bearing ahydroxy alkyl group may be succinylated with succinic anhydride and thenlinked to a cell binding agent to produce a conjugate that can becleaved by intracellular esterases to liberate free drug. {For examplessee E. Aboud-Pirak et al, Biochem Pharmacol., 38, 641-648 (1989)}.

The conjugates made by the above methods can be purified by standardcolumn chromatography or by HPLC.

Preferably conjugates between monoclonal antibodies or cell bindingagents and prodrugs of the present invention are those that are joinedvia a disulfide bond, as discussed above. Such cell binding conjugatesare prepared by known methods such as modifying monoclonal antibodieswith succinimidyl pyridyl-dithiopropionate (SPDP) {Carlsson et al, 173Biochem. J. 723-737 (1978)}. The resulting thiopyridyl group is thendisplaced by treatment with thiol containing prodrug to producedisulfide linked conjugates. Conjugates containing 1 to 10 prodrugslinked via a disulfide bridge are readily prepared by this method.Conjugation by this method is fully described in U.S. Pat. No.5,585,499, which is incorporated by reference.

In Vitro Cytotoxicity of Conjugates Between Cell Binding Agents andProdrugs of the Present Invention

Cytotoxicity of the prodrugs of the present invention and theirconjugates with cell binding agents can be measured after cleavage ofthe protecting group and conversion into the active drug. Cytotoxicityto non-adherent cell lines such as Namalwa and SW2 can be measured byback-extrapolation of cell proliferation curves as described inGoldmacher et al, 135 J. Immunol. 3648-3651 (1985). Cytotoxicity ofthese compounds to adherent cell lines such as A-375 and SCaBER can bedetermined by clonogenic assays as described in Goldmacher et al, 102 J.Cell Biol. 1312-1319 (1986).

Therapeutic Agent and Method for Inhibiting the Growth of Selected CellPopulations

The present invention also provides a therapeutic agent for inhibitingthe growth of selected cell populations comprising:

(a) a cytotoxic amount of one or more of the above-described prodrugslinked to a cell binding agent, and

(b) a pharmaceutically acceptable carrier, diluent or excipient.

Similarly, the present invention provides a method for inhibiting thegrowth of selected cell populations comprising contacting a cellpopulation or tissue suspected of containing cells from said selectedcell population with a cytotoxic amount of a cytotoxic agent comprisingone or more of the above-described prodrugs linked to a cell bindingagent.

The cytotoxic agent is prepared as described above.

Suitable pharmaceutically acceptable carriers, diluents, and excipientsare well known and can be determined by those of skill in the art as theclinical situation warrants.

Examples of suitable carriers, diluents and/or excipients include: (1)Dulbecco's phosphate buffered saline, pH about 7.4, containing about 1mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v NaCl),and (3) 5% (w/v) dextrose.

The method for inhibiting the growth of selected cell populations can bepracticed in vitro, in vivo, or ex vivo.

Examples of in vitro uses include treatments of cell cultures in orderto kill all cells except for desired variants that do not express thetarget antigen; or to kill variants that express undesired antigen.

The conditions of non-clinical in vitro use are readily determined bythe skilled artisan.

Examples of ex vivo uses include treatments of autologous bone marrowprior to their transplant into the same patient in order to killdiseased or malignant cells: treatments of bone marrow prior to theirtransplantation in order to kill competent T cells and preventgraft-versus-host-disease (GVHD).

Clinical ex vivo treatment to remove tumor cells or lymphoid cells frombone marrow prior to autologous transplantation in cancer treatment orin treatment of autoimmune disease, or to remove T cells and otherlymphoid cells from allogeneic bone marrow or tissue prior to transplantin order to prevent GVHD, can be carried out as follows. Bone marrow isharvested from the patient or other individual and then incubated inmedium containing serum to which is added the cytotoxic agent of theinvention, concentrations range from about 10 μM to 1 pM, for about 30minutes to about 48 hours at about 37° C. The exact conditions ofconcentration and time of incubation (=dose) are readily determined bythe skilled artisan. After incubation the bone marrow cells are washedwith medium containing serum and returned to the patient by i.v.infusion according to known methods. In circumstances where the patientreceives other treatment such as a course of ablative chemotherapy ortotal-body irradiation between the time of harvest of the marrow andreinfusion of the treated cells, the treated marrow cells are storedfrozen in liquid nitrogen using standard medical equipment.

For clinical in vivo use, the cytotoxic agent of the invention will besupplied as solutions that are tested for sterility and for endotoxinlevels or as a lyophilized solid that can be redisolved in sterile waterfor injection. Examples of suitable protocols of conjugateadministration are as follows. Conjugates are given weekly for 6 weeksas an i.v. bolus. Bolus doses are given in 50 to 400 ml of normal salineto which human serum albumin (e.g. 0.5 to 1 mL of a concentratedsolution of human serum albumin, 100 mg/mL) can be added. Dosages willbe about 50 μg to 10 mg/kg of body weight per week, i.v. (range of 10 μgto 100 mg/kg per injection). Six weeks after treatment, the patient mayreceive a second course of treatment. Specific clinical protocols withregard to route of administration, excipients, diluents, dosages, times,etc., can be determined by the skilled artisan as the clinical situationwarrants.

Examples of medical conditions that can be treated according to the invivo or ex vivo methods of killing selected cell populations includemalignancy of any type including, for example, cancer of the lung,breast, colon, prostate, kidney, pancreas, ovary, and lymphatic organs;melanomas; autoimmune diseases, such as systemic lupus, rheumatoidarthritis, and multiple sclerosis; graft rejections, such as renaltransplant rejection, liver transplant rejection, lung transplantrejection, cardiac transplant rejection, and bone marrow transplantrejection; graft versus host disease; viral infections, such as CMVinfection, HIV infection, AIDS, etc,; bacterial infection; and parasiteinfections, such as giardiasis, amoebiasis, schistosomiasis, and othersas determined by one skilled in the art.

EXAMPLES

The invention will now be illustrated by reference to non-limitingexamples. Unless otherwise stated, all percents, ratios, parts, etc. areby weight.

Materials and Methods

Melting points were measured using an Electrothermal apparatus and areuncorrected. NMR spectra were recorded on a Bruker AVANCE400 (400 MHz)spectrometer. Chemical shifts are reported in ppm relative to TMS as aninternal standard. Mass spectra were obtained using a Bruker Esquire3000 system. Ultraviolet spectra were recorded on a Hitachi U1200spectrophotometer. HPLC was performed using a Beckman Coulter GOLD 125system equipped with a Beckman Coulter system GOLD 168 variablewavelength detector and a Waters RADIALPAK, (a reverse phase C-18column). Thin layer chromatography was performed on Analtech GF silicagel TLC plates. Silica gel for flash column chromatography was fromBaker. Tetrahydrofuran was dried by distillation over sodium metal.Dimethylactamide and dimethylformamide were dried by distillation overcalcium hydride under reduced pressure. All other solvents used werereagent grade or HPLC grade.

The synthesis of prodrugs DC2 (2), DC3 (3) and DC4 (4) DC5 DC6 DC7 DC8(FIGS. 2-6) is described herein. DC2, DC3 and DC4 are derived from theparent drug DC1, while DC6, DC7 and DC8 can be prepared from thepegylated parent drug DC5. The prodrugs DC2 and DC3 are extremely stablein aqueous solutions, and can be converted into the parent drug DC1 byincubation in serum, plasma or with an enzyme such as carboxyl esterase.These drugs also have enhanced water solubility as compared with DC1.The prodrug DC4 is also extremely stable in aqueous solutions and alsosoluble. Incubation of DC4 with alkaline phosphatase converts it intothe parent drug DC1.

The synthetic scheme for the conversion of DC1 (1) to the prodrugs DC2(2) and DC3 (3) is shown in FIG. 4. The phenolic substituent on DC1 canbe reacted with any one of the reagents listed in FIG. 3 to give theintermediate 5. Reaction of 5 with N-methylpiperazine provides DC2.Reaction of 5 with 4-piperidino-piperidine provides DC3.

DC1 was converted to the prodrug DC4 as shown in FIG. 6. Treatment ofDC1-SMe with dibenzylphosphate and carbon tetrachloride in the presenceof base provided the intermediate 4c, while reaction of DC1-SMe withphosphorous oxychloride provide intermediate 4b. Removal of the benzylprotecting groups of 4c with hydrogen, with concomitant reduction of thedisulfide bond provided DC4. Reduction of intermediate 4b with TCEP orDTT provided DC4.

Example I Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-methylpiperazino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methyldithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC2-SMe, 2b)

To a solution of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-hydroxy-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methyldithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC1SMe) DC1-SMe (1b, 40 mg, 0.058 mmol) in THF (4 mL) was added4-nitrophenyl chloroformate (17 mg, 0.084 mmol) anddi-isopropylethylamine (DIPEA, 15 μl). The reaction mixture was stirredunder an Argon atmosphere for 3 h. Analysis by TLC showed all DC1 hadbeen consumed to form an intermediate with an Rf value of 0.45 (mobilephase of 1:2 Acetone/Toluene). The reaction mixture was treated with4-methylpiperazine (8.3 mg, 0.084 mmol), and then stirred overnightunder Argon. The mixture was then diluted with a 1:1 (v/v)} mixture ofEtOAc/fHF (15 mL) and aqueous 1 M NaH₂PO₄, pH 5.0 (5 mL). The organiclayer was separated, and the aqueous layer was extracted with EtOAc/THF(1: 1, 4×15 ml). The organic layers were combined, dried over MgSO₄,filtered, evaporated, purified by silica gel chromatography, elutingwith acetone/toluene, (3:8) and recrystallized with THF/EtOAc/Hexane toafford 40 mg (85% yield) of DC2-SMe (2b). Rf=0.31 (Acetone/foluene,3:8); mp=225° C. (dec.); ¹H NMR (DMF-d7) 11.78 (s, 1H), 11.70 (s, 1H),10.27 (s, 1H), 10.03 (s, 1H), 8.42 (d, 1H, J=1.7 Hz), 8.38 (s, 1H), 8.22(d, 1H, J=1.7 Hz), 8.12 (d, 1H, J=8.4 Hz), 7.97 (d, 1H, J=8.2 Hz), 7.72(dd, 1H, J=1.9, 8.8 Hz), 7.65 (dt, 1H, J=1.1, 7.0+7.0 Hz), 7.60 (d, 1H,J=8.9 Hz), 7.54 (t, 2H, J=7.4 8.6 Hz), 7.48 (d, 1H, J=1.4 Hz), 7.44 (dd,1H, J=1.9, 8.8 Hz) 7.36 (d, 1H, J=1.6 Hz), 5.01 (t, 1H, J=10.0 Hz), 4.84(dd, 1H, J=2.2, 10.9 Hz), 4.49 (m, 2H), 241 (dd, 1H, J=3.2, 11.3 Hz),4.10 (m, 2H), 3.82 (m, 2H), 3.22 (m, 2H), 3.13 (t, 2H, J=7.0 Hz), 296(m, 2H), 2.87 (t, 2H, J=7.1 Hz), 2.50 (s, 3H), 2.31 (s, 3H); ¹³C NMR169.52, 161.13, 160.41, 153.73, 148.70, 142.72, 134.67, 134.49, 133.67,133.36, 133.15, 132.16, 130.64, 128.41, 128.33, 128.22, 125.66, 125.59,124.06, 123.10, 122.92, 119.97, 118.31, 113.49, 112.93, 112.90, 112.11,111.66, 108.04, 106.98, 106.76, 103.80, 67.90, 67.65, 61.79, 55.82,48.15, 42.68, 37.01, 34.56, 34.32, 23.36; MS m/z+831.14 (M+Na)⁺, 833.13,832.15, 847.14 (M+K)+, 849.14, 848.14; MS m/z−807.30 (M−H)−, 808.25,809.26, 810.23.

Example II Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-methylpiperazino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC2, 2a).

A solution of tris-(2-carboxyethyl) phosphine hydrochloride (TCEP, 30mg, 0.104 mmol) in H₂O (2 mL) was adjusted to pH 7.0 with NaHCO₃ powder.To the solution was added 25 mg (0.031 mmol) of DC2-SMe (2b) in DMA (3mL). After stirring for 2 h, the pH was adjusted to between 3-4 by theaddition of a few drops of HOAc. The mixture was concentrated andpurified using preparative TLC on silica gel, eluting withacetone/toluene, 1:2) to yield 21 mg (90%) of DC2 (2a). ¹H NMR(CD₃COCD₃) 10.91 (br, 2H), 10.81 (br, 1H), 9.56 (s, 1H), 9.18 (s, 1H),8.37 (s, 1H), 8.38 (s, 1H), 8.15 (m, 1H), 8.02 (d, 1H, J=8.4 Hz), 7.94(d, 1H, J=8.6 Hz), 7.63-7.55 (m, 3H), 7.47 (m, 1H), 7.36 (dd, 1H, J=2.1,8.8 Hz), 7.29 (m, 1H), 7.21 (m, 1H), 5.44 (dd, 1H, J=2.0, 5.9 Hz), 4.85(m, 2H), 4.40 (m, 2H), 4.10 (dd, 1H, J=3.2, 11.2 Hz), 3.92 (dd, 1H,J=7.8, 11.3 Hz), 3.81 (m, 2H), 3.22 (m, 2H), 3.08 (t, 2H, J=7.0 Hz),2.87 (m, 2H), 2.82 (t, 2H, J=7.1 Hz), 2.38 (s, 3H); MS m/z+785.22(M+Na), 786.20, 787.20, 801.14 (M+K), 803.16 (M+2+K); MS m/z−762.10(M−H), 764.05, 763.08.

Example III Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-piperidino-piperidino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methydithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC3-SM2, 3b). (3b) (DC3-SMe).

To a solution of DC1-SMe, 1b, (50 mg,0.073 mmol) in THF (4 ml) was added4-nitrophenyl chloroformate (35 mg, 0.173 mmol) and DIPEA (50 μl). Afterstirring under Argon for 3 h, TLC analysis showed that all the DC1-SMehad been consumed to give an intermediate with Rf=0.45, 1:2Acetone/Toluene). The reaction mixture was treated with4-piperidino-piperidine (40 mg, 0.21 mmol), resulting in the formationof a heavy precipitate. The mixture was allowed to stir for 4 h, dilutedwith 20 ml of EtOAc/THF (1:1) and 5 ml of 1 M NaH₂PO₄, pH 4.5. Theorganic layer was separated, and the aqueous layer was extracted withEtOAc/THF (1:1, 4×15 ml). The organic layers were combined, dried overMgSO₄, filtered, evaporated, purified with Silica gel chromatography(Acetone/Foluene, 3:8) and crystallized with THF/EtOAc/Hexane to affordDC3-SMe (3b, 45 mg, 70% yield), mp=285° C. (dec.); [α]=29.7° (c 0.5 inDMF); ¹H NMR 11.93 (s, 1H), 11.76 (s, 1H), 10.44 (s, 1H), 10.09 (s, 1H),8.45 (s, 1H), 8.36 (s, 1H), 8.22 (s, 1H), 8.11 (d, 1H, J=8.3Hz), 8.00(d, 1H, J=8.4 Hz), 7.79 (dd, 1H, J=1.4, 8.6 Hz), 7.65 (t, 1H, J=7.5 Hz),7.61-7.52 (m, 3H), t.47-7.44 (m, 2H), 7.35 (d, 1H, J=1.0 Hz), 5.00 (t,1H, J=10.0 Hz), 4.83 (dd, 1H, J=1.2, 10.2 Hz), 4.67 (m, 1H), 4.51 (m,1H), 4.30 (m, 1H), 4.21 (dd, 1H, J=3.1, 11.1 Hz), 4.11 (m, 1H), 3.40 (m,2H), 3.12 (t, 2H, J=7.0 Hz), 3.10 (m, 2H), 2.95 (m, 1H), 2.87 (t, 2H,J=7.1 Hz), 2.49 (s, 3H), 2.39 (m, 2H), 2.12 (m, 2H), 2.02-1.60 (m, 10H);¹³C NMR 169.52, 162.90, 161.15, 160.34, 153.55, 148.47, 142.66, 134.66,134.48, 133.76, 133.35, 133.29, 132.08, 130.63, 128.37, 128.22, 125.69,125.52, 123.47, 123.11, 120.01, 118.01, 113.43, 112.89, 112.08, 111.72,63.44, 60.08, 56.01, 50.00, 48.08, 42.62, 37.01, 34.07, 24.10, 24.08,23.08, 22.78; MS m/z+878.24 (M+H)⁺, 880.24, 879.25, 880.24; MSm/z−876.40 (M−H)⁻, 878.34, 877.37, 879.35.

Example IV Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-piperidino-piperidino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC3, 3a). (3a) (DC3).

A solution of TCEP (15.2 mg, 0.053 mmol) in H₂O (0.7 mL) was adjusted topH 7.0 by the addition of 13.5 mg of NaHCO₃ powder. To the solution wasadded DC3-SMe (3b, 8.2 mg, 0.0093 mmol) in DMA (2 mL), and the reactionmixture was stirred for 2 h. A few drops of HOAc was then added toadjust the pH to between 3-4. The mixture was concentrated and purifiedusing preparative TLC on silica gel, eluting with acetone/toluene, (1:2)to yield 7 mg of DC3 (3a). MS m/z+854.31 (M+Na)+, 855.31, 856.32,857.31.

Example V Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-(phosphonoxy)-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methyldithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide(4b) (DC4-SMe)

A solution of DC1-SMe (1b, 50 mg, 0.073 mmol) in a mixture of THF (5ml), CH₃CN (4 ml) and DMA (0.5 ml) was stirred under an atmosphere ofArgon. To the mixture were sequentially added POCl₃ (80 μL), DIPEA (150μL) and DMAP (3 mg). After stirring for 2 h, both TLC and HPLC analysesshowed that the DC1-SMe had been completely consumed. Aqueous 1.0 MNaH₂PO₄, pH 4.0 (2 ml) was added, and the mixture was stirred overnight.The mixture was further acidified with H₃PO₄ to pH 2.0, saturated withNaCl, and extracted with THF/EtOAc (1:1, 6×15 ml). The organic layer wasseparated, concentrated and the residue was recrystallized withTHF/H₂O/CH₃OH to afford 47 mg (84%) of the title compound (DC4-SMe). ¹HNMR (DMF-d7) 11.77 (s, 1H), 11.70 (s, 1H), 10.26 (s, 1H), 10.02 (s, 1H),8.74 (s, 1H), 8.42 (s, 1H), 8.30 (d, 1H, J=7.6 Hz), 8.22 (s, 1H), 7.72(d, 1H, J=8.3 Hz), 7.59 (m, 2H), 7.55-7.43 (m, 4H), 7.33 (s, 1H), 4.96(t, 1H, J=9.8 Hz), 4.81 (d, 1H, J=10.2 Hz), 4.42 (m, 1H), 4.18 (m, 1H),4.05 (dd, 1H, J=7.8, 11.0 Hz), 3.11 (t, 2H, J=7.0 Hz), 2.87 (t, 2H,J=7.1 Hz), 2.49 (s, 3H); ¹³C NMR 169.96, 161.45, 160.85, 143.10, 135.12,134.93, 134.15, 133.81, 133.58, 132.80, 131.60, 128.86, 128.78, 128.02,124.50, 124.05, 120.22, 118.61, 113.97, 113.40, 113.32, 112.57, 108.02,104.27, 56.18, 48.60, 43.21, 37.47, 34.49, 23.53; ³¹P NMR −3.37; MS m/z762.77 (M−H)⁻, 764.80, 763.76.

Example VI Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-(phosphonoxy)-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide(4a) (DC4)

A solution of TCEP (30 mg, 0.104 mmol) in 2 ml of H₂0 was adjusted to pH6.5-7.0 by the addition of NaHCO₃ powder. To the solution was addedDC4-SMe (26 mg, 0.034 mmol) in 3 ml of DMA/H₂O (1:1). After beingstirred for 2 h under Argon, a few drops of 10% H₃PO₄ was added to reachpH 2.0. The mixture was then extracted with DMA/EtOAc (1:5, 6×10 ml).The organic layers were combined, evaporated and purified by preparativeHPLC, using a C18 column, 20×250 mm, flow rate=8.0 ml/min., mobilephase: A: 0.01% HOAc in H₂O, B: 2% DMA in CH₃CN; time table: 0-10′, 5%B; to 20′, 20% B; to 50′: 50% B. The DC4 peak eluted between 30-38 min.The fractions containing DC4 were pooled, concentrated and dried undervacuum to yield 22 mg (89%) of the title compound 4a. ¹H NMR (DMF-d7)11.76 (s, 1H), 11.69 (s, 1H), 10.26 (s, 1H), 10.02 (s, 1H), 8.78 (s,1H), 8.41 (s, 1H), 8.29 (d, 1H, J=9.2 Hz), 4.80 (d, 1H), 7.79 (d, 1H,J=5.1 Hz), 7.60-7.43 (m, 6H), 7.27 (s, 1H), 4.96 (t, 1H, J=9.2 Hz), 4.80(d, 1H, J=10.6 Hz), 4.42 (m, 1H), 4.23 (dd, 1H, J=2.2, 9.4 Hz), 4.06(dd, 1H, J=7.4, 10.6 Hz), 3.12 (t, 2H, J=7.1 Hz), 2.88 (t, 2H, J=7.1Hz); MS m/z 716.16 (M−H)⁻, 717.12, 718.12.

Alternatively, to a solution of DTT (20 mg) in a mixture of acetone (3mL) and 50 mM NaH₂PO₄ buffer (3 mL), pH 7.0 was added DC4SMe (22 mg,0.028 mmol). After stirring under Argon for 4 h, a few drops of 5% H₃PO₄was added to pH 3.0. The mixture was concentrated and purified on a C18column (1.0×12 cm) eluted with 100% water to 50% water in acetone. Thefractions were pooled and evaporated to dryness to afford 18 mg (90%) ofDC4 (4a). MS m/z−716.30, 718.30, 717.30.

Example VII Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-(dibenzylphosphonoxy)-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methyldithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide(4c) (DC4-SMe dibenzylphosphate)

To a solution of DC1-SMe (50 mg, 0.073 mmol) in 10 ml of THF/CH₃CN (1:1)under argon were sequentially added CCl₄ (100 μL, 1.036 mmol), DIPEA (55μL, 0.316 mmol), dibenzylphosphite (100 μL, 0.452 mmol) and DMAP (0.2mg, 0.0016 mmol). After stirring overnight under Argon, the reactionappeared complete by TLC analysis, with a new product being formed withan Rf=0.37 in acetone/toluene 1:2). The mixture was diluted with 5 ml of1.0 M NaH₂PO₄, pH 4.0, and EtOAc (10 mL). The organic layer wasseparated and the aqueous solution was extracted with THF/EtOAc (1:1,4×15 ml). The organic layers were combined, dried over MgSO4, filtered,evaporated and purified by silica gel chromatography, eluting withacetone/toluene (3:7), to afford 62 mg (89%) of the title compound 4c.¹H NMR (DMF-d7) 11.84 (s, 1H), 11.74 (s, 1H), 10.31 (s, 1H), 10.04 (s,1H), 8.77 (s, 1H), 8.44 (s, 1H), 8.10 (t, 2H, J=7.3 Hz), 7.74 (dd, 1H,J=1.7, 8.8 Hz), 7.66-7.61 (m, 2H), 7.55-7.29 (m, 15H), 5.37 (t, 4H,J=7.5 Hz), 5.01 (m, 2H), 4.84 (dd, 1H, J=1.9, 10.9 Hz), 4.51 (m, 1H),4.20 (dd, 1H, J=3.2, 10.9 Hz), 4.11 (dd, 1H, J=6.9, 11.1 Hz), 3.13 (t,2H, J=7.2 Hz), 2.87 (t, 2H, J=7.1 Hz), 2.50 (s, 3H); ¹³C NMR 169.52,161.18, 160.41, 147.88, 147.20, 136.74, 136.67, 134.68, 134.56, 133.70,133.36, 133.20, 132.11, 130.83, 129.25, 129.23, 129.21, 129.19, 128.84,128.78, 128.62, 128.42, 128.32, 127.90, 124.28, 124.22, 123.96, 123.33,122.87, 120.03, 118.32, 113.51, 112.94, 112.12, 108.42, 106.87, 70.75,70.69, 67.91, 55.90, 47.96, 42.59, 37.02, 34.04, 23.08; ³¹P NMR −4.49;MS m/z 966.17 (M+Na)⁺, 968.14 (M+2+Na), 967.17.

Conversion to DC4:

A flask was charged with 4c(20 mg, 0.021 mmol), and treated with Pd/C(15 mg), glacial acetic acid (100 μl) and DMA (4 ml). The system wasevacuated with vacuum suction, and then stirred under hydrogen through ahydrogen-filled balloon overnight. The catalyst was removed byfiltration and the solvent was evaporated, and the residue was purifiedby preparative HPLC as above described to yield 6 mg (39%) of DC4 (4a).MS m/z 716.48 (M−H)⁻, 717.48, 718.50.

Example VIII Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-hydroxy-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-nitro-1H-indole-2-carboxamide(DC0, 10a).

To a solution of5-hydroxy-3-amino-1-[S]-(chloromethyl)-1,2-dihydro-3H-benz(e)indole,hydrochloride salt [seco-(−) CBI, 20 mg, 0.72 mmol] and5-[5′-nitroindol-2′-yl-carbonyl amino]indole-2-carboxylic acid (9, 25mg, 0.068 mmol) in DMA (3 mL) was added EDC (40 mg, 0.20 mmol) underArgon. After stirring overnight, a few drops of 50% HOAc were added andthe mixture was evaporated to dryness and purified by preparative silicagel TLC chromatography (40% acetone in toluene) to afford 25 mg of DCO(10a). ¹H NMR (DMF-d₇) 12.54 (s, 1H), 11.73 (s, 1H), 10.60 (s, 1H),10.58 (s, 1H), 8.80 (d, 1H, J=2.3 Hz), 8.42 (d, 1H, J=1.9 Hz), 8.25 (d,1H, J=8.5 Hz), 8.19 (dd, 1H, J=2.1, 9.1 Hz), 8.09 (br, H), 7.95 (d, 1H,J=8.3 Hz), 7.82 (d, 1H, J=1.5 Hz), 7.79 (d, 1H, J=9.1 Hz), 7.74 (dd, 1H,J=2.0, 8.9 Hz), 7.62 (d, 1H, J=8.8 Hz), 7.58 (dt, 1H, J=1.7, 7.0+7.0Hz), 7.42 (dt, 1H, J=1.2, 7.0+7.0 Hz), 7.33 (d, 1H, J=1.7 Hz), 4.91 (t,1H, J=11.0 Hz), 4.77 (dd, 1H, J=2.1, 11.1 Hz), 4.33 (m, 1H), 4.13 (dd1H, J=3.1, 11.1 Hz), 3.97 (dd, 1H, J=7.9, 11.1 Hz); 1³C NMR 163.35,161.48, 160.05, 155.79, 142.98, 137.18, 135.03, 133.22, 133.16, 131.50,128.85, 128.45, 128.11, 124.62, 124.02, 123.76, 120.33, 119.36, 118.70,116.45, 114.00, 113.08, 106.97, 105.02, 101.53; MS m/z 602.96 (M+Na)⁺,604.78, 603.81, 618.64 (M+K)⁺, 620.48.

Example IX Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-hydroxy-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methyldithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide,DC1SMe (1b)

A flask was charged with 10a (10 mg, 0.017 mmol), Pd/C (10 mg), HCl(conc. 3 μl) and DMA (2.5 ml). After the air was evacuated, hydrogen wasconducted by hydrogen balloon overnight. The catalyst was removed byfiltration and the solvent was evaporated to give 10b as a brown solid.The solid compound was used directly without further purification.

To 10b in DMA (2 mL) was added 3-(methyldithio)propionic acid (5 mg,0.032 mmol) and EDC (15 mg, 0.078 mmol) under Argon. After stirringovernight, two drops of 50% HOAc were added, and the mixture wasevaporated to dryness and purified by preparative silica gel TLC (40%acetone in toluene) to afford 6 mg of DC1-SMe (1b) Rf=0.40(3:7Acetone/Toluene); ¹H NMR (CD₃COCD₃) 10.91 (s, 1H), 10.88 (s, 1H), 9.64(s, 1H), 9.56 (s, 1H), 9.27 (s, 1H), 8.35 (d, 1H, J=1.9 Hz), 8.25 (d,1H, J=8.0 Hz), 8.17 (d, 1H, J=1.9 Hz), 8.07 (s, 1H), 7.88 (d, 1H, J=8.3Hz), 7.64 (dd, 1H, J=2.0, 8.1 Hz), 7.58-7.50 (m, 3H), 7.38-7.35 (m, 2H),7.31 (d, 1H, J=1.7 Hz), 7.26 (d, 1H, J=1.7 Hz), 4.86 (dd, 1H, J=8.7,11.0 Hz), 4.80 (dd, 1H, J=2.3, 10.9 Hz), 4.30 (m, 1H), 4.07 (dd, 1H,J=3.1, 11.0 Hz), 3.83 (dd, 1H, J=8.4, 11.2 Hz), 3.09 (t, 2H, J=7.1 Hz),2.83 (t, 2H, J=7.1 Hz), 2.45 (s, 3H); ¹³C NMR 169.56, 161.10, 160.43,155.13, 143.50, 134.78, 134.46, 133.55, 133.34, 133.03, 132.57, 131.21,128.80, 128.69, 128.21, 124.22, 124.02, 123.53, 123.44, 120.16, 118.79,116.45, 113.91113.02, 112.95, 112.73, 106.78, 103.72, 101.63, 56.01,47.73, 43.10, 37.25, 34.01, 23.00; MS m/z 706.71 (M+Na)⁺, 708.58,707.71, 722.34 (M+K)⁺, 724.42.

Example X Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-hydroxy-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(15″-methyldithio4″,7″,10″,13″-tetraoxapentadecyl-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC5-SMe)

To a solution of 10b (50 mg, 0.091 mmol) in DMA (5 mL) was added15″-methyldithio-4″,7″,10″,13″-tetraoxapentadecanoic acid (33 mg 0.100mmol) and EDC (88 mg,0.459 mmol) under Ar. After being stirredovernight, two drops of 50% HOAc were added to the mixture and themixture was evaporated to dryness, purified by Silica gel chromatography(30% acetone in toluene) to afford DC5-SMe

Certain patents and printed publications have been referred to in thepresent disclosure, the teachings of which are hereby each incorporatedin their respective entireties by reference.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one of skill in theart that various changes and modifications can be made thereto withoutdeparting from the spirit and scope thereof.

1. A prodrug of an analog of CC-1065, having three subunits linked byamide bonds, wherein the first subunit is a seco-CBI(cyclopropabenzindole) molecule represented by the following formula:

wherein X is halogen, and wherein the second and third subunits togetherare represented by the formula (II):

wherein R represents a linking group that enables linkage of saidprodrug to a cell binding agent; R₁-R₆ are each independently hydrogen,C₁-C₃ linear alkyl, methoxy, hydroxyl, primary amino, secondary amino,tertiary amino, or amido; and R₇ is piperazine carbamate,4-piperidino-piperidino carbamate or phosphate, and wherein the compoundof formula (I) is covalently bonded the compound of formula (II) via thesecondary amino group of the pyrrole moiety of the first subunit to theC-2 carboxyl of the second subunit.
 2. The prodrug of claim 1, wherein Rcomprises a thiol or a disulfide bond.
 3. The prodrug of claim 1,wherein the linking group is selected from the group consisting ofNHCO(CH₂)_(m)SZ, NHCOC₆H₄(CH₂)_(m)SZ, NHCOC₆H₄O(CH₂)_(m)SZ,NHCO(CH₂)_(m)(OCH₂CH₂)_(n)SZ, NHCOC₆H₄(CH₂)_(m)(OCH₂CH₂)_(n)SZ, andNHCOC₆H₄O(CH₂)_(m)(OCH₂CH₂)_(n)SZ wherein: Z represents H or SR₈,wherein R₈ represents methyl, linear alkyl, branched alkyl, cyclicalkyl, simple or substituted aryl or heterocyclic selected from thegroup consisting of furyl, pyrrollyl, pyridyl, and thiophene, mrepresents an integer of 1 to 10, and n represents an integer of 4 to1000.
 4. The prodrug of claim 1, wherein the linking group is selectedfrom the group consisting of NHCO(CH₂)_(m)(OCH₂CH₂)_(n)SZ,NHCOC₆H₄(CH₂)_(m)(OCH₂CH₂)_(n)SZ, and NHCOC₆H₄O(CH₂)_(m)(OCH₂CH₂)_(n)SZwherein: Z represents H or SR₈, wherein R₈ represents methyl, linearalkyl, branched alkyl, cyclic alkyl, simple or substituted aryl orheterocyclic selected from the group consisting of furyl, pyrrollyl,pyridyl, and thiophene, m represents an integer of 1 to 10, and nrepresents an integer from 2 to
 1000. 5. The prodrug of claim 3, whereinthe linking group is selected from the group consisting of NHCO(CH₂)₂SH,NHCO(CH₂)₂SSCH₃, NHCO(CH₂)₂(OCH₂CH₂)_(n)SH andNHCO(CH₂)₂(OCH₂CH₂)_(n)SSCH₃.
 6. The prodrug of claim 1, wherein thefirst subunit is represented by formula (I):